The present invention relates to compositions and methods for treating sperm for the purpose of modifying sperm function and the gender ratio in offspring of mammalian species. The invention is further directed to a method of using the composition to modify the functionality of mammalian sperm in general and more specifically, to increase sperm fertility for the purpose of enhancing conception. In addition, the invention can be used to modify the fertility of X-chromosome and Y-chromosome bearing sperm and using said sperm in the reproduction processes of artificial insemination (AI), in vitro fertilization (IVF), and embryo transfer (ET) for the purpose of modifying gender in mammals.
It is well documented in mammalian species that the X-chromosome contains a unique set of genes which are highly conserved across mammals as well as other vertebrates. One of the X-linked genes codes for the ubiquitous enzyme, glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is a pivotal enzyme in glucose metabolism and is the primary regulator of the hexose-mono phosphate shunt (HMS), also known as the pentose phosphate shunt (PPP). The main function of the HMS is to produce NADPH, which is necessary for reduction-oxidation reactions and to form ribose-5-phosphate for nucleic acid synthesis.
G6PDH also play an important role in glucose oxidation via glycolysis, a primary source of cellular energy. Glucose metabolism is implicated in the fertilization process in many species (mouse, Hoppe, 1976; rat, Niwa and Iritani, 1978; human, Mahadevan et al., 1997, incorporated herein by reference). It is well accepted that glucose metabolism through glycolysis provides energy to sperm. However, the role of glucose metabolism through the hexose-monophosphate shunt (HMS) in spermatozoa is not understood. The existence of an HMS pathway in mouse (Setchell et al, 1969, incorporated herein by reference) and human sperm (Aitken et al, 1997 incorporated herein by reference) has been documented, but not in other species including ram or bull sperm. The techniques used for these early studies have come into question and the evidence does not prove or disprove the existence of the HMS in sheep or cattle sperm.
The implications of a functioning HMS in sperm, as found in human and mouse sperm, however does suggests that it is implicated in sperm function. Since NADPH metabolism has been implicated in sperm motility and fertilization, it has been suggested that the HMS is a key metabolic pathway in sperm capacitation, the acrosome reaction, and oocyte fusion (Urner, F. and Sakkas, D., 1999, incorporated herein by reference).
While investigating the role of the X-linked enzyme, G6PDH, and the HMS as a method to sex mouse embryos, I first used the phenoxazine compound, brilliant cresol blue (BCB) in living embryos to detect and semi-quantify G6PDH activity (Williams, 1986, incorporated herein by reference) and successfully transplant these embryo to produce normal living offspring. This study demonstrated that BCB and its metabolites were relatively non-toxic in the early-staged mouse embryos. My studies with bovine embryos revealed similar results. While investigating the HMS in bovine oocytes, Tiffen et al., 1991, (incorporated herein by reference) demonstrated that the phenoxazine/phenothiazine class of compounds could be used to increase glucose oxidation specifically through the Hexose Monophosphate Shunt (HMS). They also saw an increased level of glucose metabolism through glycolysis. These authors documented that BCB causes a 15-fold increase in oxidation of glucose through the HMS and a somewhat less increase through glycolysis.
It is well documented that early staged female embryos exhibit preferential metabolism of glucose via the HMS and that male embryos preferential use glycolysis (Tiffin et al., 1991, Kimura et al, 2005, incorporated herein by reference). This is due to the presence of two X-chromosomes and elevated levels of G6PDH in the female. Male embryos have a single X-chromosome and low levels of G6PDH.
My postulate is that the sexual dimorphism in glucose metabolism extends to the sperm and spermatocytes. This would mean that levels of the X-linked enzyme, G6PDH, are very low in the Y-chromosome bearing sperm due to the absence of the X-chromosome. Given this assumption, the Y-chromosome bearing sperm (similar to male embryo) cannot oxidize glucose or G-6-P through the HMS, but oxidizes large amounts of G-6-P through glycolysis. Since phenoxazine catalyzes glycolysis it is hypothesized that the addition of G-6-P and phenoxazine further amplifies glycolysis and leads to increased ATP production, capacitation, motility, and the observed increase in fertility of the Y-chromosome bearing sperm.
In the X-chromosome bearing sperm, assumed to have high levels of G6PDH due to the presence of the X-chromosome, G-6-P would be metabolized through the HMS. However, when exposed to the composition of this invention, the oxidation of G-6-P appears inhibited and results in reduced fertility levels and fewer female offspring. This inhibition through the HMS is likely due to a modified redox potential caused by the oxidant chromo-phenoxazine and the absence of an electron transfer agent such as NADPH oxioreductase resulting in a rate limiting turnover (oxidation) of endogenous NADPH. Without the reductive power of NADPH, the chromo-phenoxazine remains in the oxidative state and quenches the HMS. In the presence of phenoxazine (without a NADPH oxioreductase or a similar electron transfer agent), oxidation of G-6-P is shutdown resulting in an inhibition of ATP production and a loss of sperm motility and sperm function specific to the X-chromosome bearing (female) sperm.
Another factor influencing the X-chromosome bearing (female) sperms preferential oxidization of G-6-P through the HMS pathway is the major cofactor NADP+ which also regulates the pathway. This cofactor is needed in the first and third steps of the HMS pathway. Without the replenishment of NADP+ by the recycling of NADPH (due the oxidative state of the cell) the pathway would quickly become inhibited and eventually quench resulting in a loss in needed energy precursors. This could contribute to the X-chromosome bearing (female) sperm losing function, motility, and fertilization capacity.
Another observation I have made is that sperm exposed to phenoxazine compounds without added substrates quickly lose sperm motility and subsequent fertility. This loss of motility is likely due to a quenching of energy metabolism of the cell including glycolysis and the HMS, but more importantly the loss of ATP production via quenching of oxidative phosphorylation and respiratory potential. The result is little or no motility and a potential loss of fertilization capacity regardless of sperm gender. This observation points to the role of chromo-phenoxazine in quenching or slowing the respiratory cycle which is dependent upon large amounts of reducing power in the form NADH.
Patent documents of interest concerning methods to preferentially modify sperm function include: U.S. Pat. Nos. 4,191,749, 4,191,749, 4,999,283, 4,788,984, 6,627,655, and 20070166694
Patent documents of interest concerning the use of phenoxazine BCB in living tissue are limited to in vitro assays, dye indicators, and staining methodology. These include: U.S. Pat. Nos. 6,790,411, 6,867,015, 6,967,015, 6,420,128, and 4,622,395.
Patent documents of interest related to methods of modifying sex ratio in mammals by cell sorting technology include: U.S. Pat. Nos. 6,524,860, 6,372,422, 6,149,867, 6,071,689, and 5,135,759.
Patent documents of interest related to modifying sex ratio in mammals by antigen or antibody sorting include: U.S. Pat. Nos. 6,489,092, 6,153,373, 5,660,997, and 5,439,362.